Esculetin triggers ferroptosis via inhibition of Nrf2-xCT/GPx4 axis in hepatocellular carcinoma

Esculetin is a natural dihydroxy coumarin derived from Chinese herbal medicine Cortex Fraxini and has shown significant pharmacological activities, including anticancer properties. Ferroptosis, an iron-dependent form of regulated cell death, has garnered considerable attention due to its lethal effect on tumor cells. However, the exact role of ferroptosis in esculetin-mediated anti-hepatocellular carcinoma (HCC) effects remains poorly understood. In this study, we observed that esculetin effectively inhibited the growth of HCC cells both in vitro and in vivo. Importantly, esculetin promoted the accumulation of intracellular Fe^{2 +} , leading to an increase in reactive oxygen species (ROS) production through the Fenton reaction. This event subsequently induced lipid peroxidation (LPO) and triggered ferroptosis within the HCC cells. The occurrence of ferroptosis was confirmed by the elevation of malondialdehyde (MDA) levels, the depletion of glutathione peroxidase (GSH-Px) activity, and the disruption of mitochondrial morphology. Notably, the inhibitor of ferroptosis, ferrostatin-1 (Fer-1), inhibited the anti-tumor effect of esculetin in HCC cells. Furthermore, our findings revealed that esculetin inhibited the Nrf2-xCT/GPx4 axis signaling in HCC cells. Overexpression of Nrf2, in turn, upregulated the expression of downstream SLC7A11 and GPX4, consequently alleviating esculetin-induced ferroptosis. In conclusion, our results suggest that esculetin exerts an anti-HCC effect by inhibiting the activity of the Nrf2-xCT/GPx4 axis, thereby triggering ferroptosis in HCC cells. These findings may contribute to the potential clinical use of esculetin as a candidate for HCC treatment.